news
RESEARCH PROFILE CE/MS for quantitative metabolome analysis Many scientists believe one of the keys to understanding human diseases lies in the metabolome. A cell contains more than 1000 metabolites, which play a major role in cell phenotype and gene expression. But their high polarity, nonvolatility, and poor detectability have made detection difficult. Previous analytical methods such as GC/MS, NMR, FTICR MS, and ESI-MS have failed to quantitatively pinpoint them. As reported in this issue of Journal of Proteome Research (pp 488–494), Tomoyoshi Soga and colleagues at Keio University (Japan) believe they can succeed where other researchers have failed by looking at metabolites using CE/MS. Previous methods either lacked quantification and sensitivity, needed chemical derivatization, or couldn’t separate multiple isomers, says Soga. “Moreover, MS infusion techniques have recently become popular, but [those] techniques cannot do quantitative analysis because of ion suppression effects,” states Soga. “For example, if a sample is diluted at 1:1 and 1:10 and analyzed by MS infusion approaches, the obtained MS patterns are different.” On the other hand, Soga and colleagues detected 1692 metabolites, and, with the help of bioinformatics, they identified 150 and assigned 83. The methods had detection limits as low as 40 zmol for adenine and 350 amol for Glu. And some of the findings from studies of sporulation challenge current ideas about predicting gene expression from analysis of the transcriptome. Soga and his colleagues are familiar with using CE/MS for metabolite determination (Anal. Chem. 2002, 74, 2233–2239). To overcome the individual limitations of the previous detection methods, the researchers worked out three CE/MS methods to concurrently probe for cationic metabolites, anionic metabolites, and nucleotides from the bacteria Bacillus subtilis. “With this approach, every charged
© 2003 American Chemical Society
metabolite can be simultaneously determined by only three conditions,” says Soga. To observe the maximum number of cationic species, the researchers used a very low pH electrolyte to confer a positive charge to the cations, which made them more susceptible to MS analysis. To measure the anionic metabolites, the researchers reversed the electroosmotic flow using a positively charged, polymercoated capillary. A similar method was utilized for the nucleotide analysis except that a noncharged polymer and pressure were added (Anal. Chem. 2002, 74, 6224– 6229). The researchers say another benefit to their three-pronged approach is that metabolites can be analyzed on a smaller scale compared with conventional largescale probes. “With our CE/MS [method], researchers need only 30 nL of sample per run,” says Soga, which allows several hundred analyses from a 10-µL sample. Analyzing such small volumes also means that samples can be highly concentrated, which increases the detection sensitivity, adds Soga. The researchers scanned for metabolites over a broad range of 70–1027 m/z. Because of the low concentration of metabolites in B. subtilis cells, they limited the scan range to 30 m/z to obtain the greatest sensitivity and analyzed each sample more than 30 times. They positively identified 150 metabolites. To identify unknowns, the researchers first estimated the number of carbons on the
“CE”ing all angles. Schematic depicting setups for simultaneous detection of (a) cationic metabolites, (b) anionic metabolites, and (c) nucleotides and CoA compounds using CE/MS.
basis of measured molecular weight and 13C contributions, which led to the chemical formula. Candidates were then selected from the LIGAND database of known metabolites, and a final formula of the chemical compound was then predicted after charge, electrophoretic mobility, and isotopic contributions were considered. Using this method, the researchers assigned a total of 60 compounds and 23 original formulas. Identification of previously unknown metabolites was not the only success of Soga’s method. It also allowed the researchers to survey significant changes in B. subtilis cells during sporulation. The most noteworthy changes were in the tricarboxylic acid (TCA) cycles. Metabolites also were analyzed at various stages before and during spore formation. The results showed that levels of most metabolites in the glycolytic, pentose phosphate, and TCA pathways decreased substantially during sporulation. Several previously uncharacterized compounds—including those tentatively determined to be L-homocitrulline, alanyl-L-lysine, and sedoheptulose 7phosphate—varied, which suggests that these compounds may play previously unsuspected roles in sporulation. As expected, the TCA-cycle intermediates cis-aconitate and isocitrate accumulated at the start of sporulation and later decreased, while acetyl and succinyl CoA increased. What didn’t fit quite as well was the observation that cis-aconitate, isocitrate, CoA, acetyl and succinyl CoA, Lys, and -Ala levels increased even though their gene expression levels decreased. The researchers suggest that this finding challenges the notion that gene expression analysis accurately predicts metabolite levels. The researchers say the next step is to use other methods, perhaps CE/ time-of-flight MS or CE/MS/MS, to determine the chemical formulas of and structural information about unknown compounds. They also suggest that metabolome analysis might be combined with transcriptome and proteome analysis to explore uncharacterized or poorly characterized biological processes.
Journal of Proteome Research • Vol. 2, No. 5, 2003
463
MEETING NEWS Katie Cottingham reports from the 51st American Society for Mass Spectrometry Conference —Montreal, Canada
SCOTT MCLUCKEY, PURDUE UNIVERSITY
—Wilder D. Smith
Top-down MS. Singly charged species in an ion trap.
“Top-down” MS without a magnet? According to conventional wisdom, you need an expensive FT mass spectrometer equipped with a powerful magnet to perform top-down MS, but this isn’t the case anymore. Among the new approaches coming on the scene are two top-down methods that use less costly ion trap and TOF-TOF mass spectrometers. FTMS can resolve isotopic peaks associated with protein fragments that have the same nominal m/z after electrospray ionization, but Scott McLuckey of Purdue University says, “The resolution of our device [an ion trap] isn’t high enough to do that, so instead we manipulate the charges by taking multiply charged ions and converting them to singly charged
ions.” To do this, McLuckey and his colleagues added an atmospheric sampling glow discharge ionization (ASGDI) source to an ion trap. ASGDI produces negatively charged ions that react with multiply charged positive product ions in the trap. Every product ion then becomes singly charged, which makes the job of deducing masses from the measured m/z values much easier. Detlev Suckau and Anja Resemann of Bruker Daltonik (Germany) address the problem of analyzing the amino- and carboxy-termini of proteins with a new topdown method called T3 sequencing. They ionize a whole protein and fragment it twice using a TOF-TOF mass spectrometer in a “pseudo MS3 experiment” to ana-
lyze modified terminal ends and obtain structural information. “The idea came up when we realized that we have two types of fragmentations that we can use on the MALDI TOFTOF: a fast process on the nanosecond timescale inside the source [in-source decay (ISD)] and a slower fragmentation process occurring after the ions left the source,” says Suckau. ISD is used to obtain a protein’s terminal peptides, which are then analyzed on the TOF-TOF. With current methods, termini are difficult to study, particularly if they are posttranslationally modified, but Suckau says that this technique allows researchers to see “whether at the termini unexpected things, such as modifications, are happening.” He says T3 sequencing can be used as a fast quality control protocol in protein purification and recombinant protein work, where such tools are “badly needed”. With T3 sequencing, for instance, researchers can put the human protein they are producing in yeast into a TOF-TOF and make sure that it is still being correctly modified, or they can check a protein to see if it has the right modification on it before putting it on a chip.
Journal of Proteome Research • Vol. 2, No. 5, 2003
465
